WO 00/32767 describes soluble Fc receptors (FcRs) which are composed of only the extracellular part of the receptor and are not glycosylated. Due to the absence of the transmembrane domain and of the signal peptide, these proteins are present in a soluble form and not bound to cells. Furthermore the FcRs described in WO 00/32767 can be produced recombinantly and have been suggested for the treatment of autoimmune diseases due to their ability to bind the Fc part of antibodies without interfering with other components of the immune system. WO 00/32767 additionally describes the crystal structure of certain FcRs and the possibility of finding substances that inhibit the interaction of IgG with FcRs with the aid of these crystal structures. The elucidation of the crystal structure enables finding such inhibitors by screening the databases using available computer programs.
The invention which was defined in WO 03/043648 further developed the findings of WO 00/32767 and provides treatment methods especially for diseases like multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) and also for diseases with an elevated level of natural killer cells. When said receptors were produced recombinantly in prokaryotes and therefore were unglycosylated the inventors of WO 03/043648 surprisingly found that although the unglycosylated proteins were expected to be poorly soluble, the receptors could be purified with high concentrations of FcγR in a soluble form.
WO 03/043648 and other publication documents, that FcRs play an important role in defense reactions of the immune system. When pathogens have entered the blood circulation they are bound by immunoglobulins, also known as antibodies. Since the immune response to a pathogen is poylclonal, a multitude of antibodies bind to a pathogen, leading to the formation of a so called immune-complex (IC). ICs are subsequently phagocytised by specialized effector cells (e.g. phagocytes or macrophages) of the immune system and thus removed from the circulation. The phagocytosis is mediated by the binding of the Fc-part of the antibodies, forming the IC together with the pathogen, to FcRs on the aforementioned effector cells. Other effector cells of the immune system, such as natural killer cells, eosinophils and mast cells also carry FcRs on their surface which upon binding of immune complexes release stored mediators such as growth factors or toxins that support the immune response.
The FcRs of these effector cells also function as signal-transucing molecules that specifically bind immunoglobulins of various isotypes during the humoral immune response. In addition FcRs expressed on natural killer cells play a fundamental role in the destruction of antibody-coated target cells (“antibody-dependent cell-mediated cytotoxicity”, ADCC).
However, in addition to the positive effects of FcRs in the defense against pathogens, overshooting reactions caused by the presence of auto-antibodies in patients may also occur which result in an undesired stimulation of the immune system which manifests itself especially as inflammatory or autoimmune diseases. Such immune reactions directed against the body's own substances remain a major medical problem and although there are approaches for treating them, these approaches are not equally effective in every patient.
All members of the FcγR-family are integral membrane glycoproteins, possessing extracellular domains related to a C2-set of immunoglobulin-related domains, having a single membrane spanning domain and an intracytoplasmic domain of variable length. There are three known Fcγ receptor forms, designated FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). This invention specifically focuses on FcγRII (CD32).
FcγRII proteins are 40 KDa integral membrane glycoproteins which bind only bind the complexed IgG. These receptors are the most widely expressed FcγRs, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. There are three human FcγRII genes (FcγRII-a, FcγRII-b, FcγRII-c), all of which bind IgG in aggregates or immune complexes.
Inflammation is a process by which the body's white blood cells react to infection by foreign substances, such as bacteria and viruses. It is usually characterized by pain, swelling, warmth and redness of the affected tissue. Effector substances known as cytokines and prostaglandins control this process, and are released in an ordered and self-limiting cascade into the blood or affected tissues. The release of such effector substances increases the blood flow to the area of injury or infection. Some of the effector substances cause a leak of fluid into the tissues, resulting in swelling. This protective process may stimulate nerves and cause pain. These changes, when occurring for a limited period in the relevant area, work to the benefit of the body.
In autoimmune diseases the patient's immune system has lost the ability to discriminate between body-own (“self”) and foreign proteins. In consequence, antibodies are generated that recognize “self”-proteins and form immune complexes which continuously activate the immune system because the “self”-protein is permanently produced and recognized as foreign. This chronic condition can persist for years leading in the end to severe organ damage and possibly to the death of the patient. There are many different autoimmune disorders which affect the body in various ways. For example, the brain is affected in individuals with multiple sclerosis, the gut is affected in individuals having Crohn's disease, and the synovium, bone and cartilage of various joints are affected in individuals suffering from rheumatoid arthritis. As autoimmune disorders progress destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function may result. The autoimmune disorder may affect a single organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include red blood cells, blood vessels, connective tissues, endocrine glands (e.g., the thyroid or pancreas), muscles, joints, and the skin.
Examples of inflammatory and/or autoimmune disorders include, but are not limited to, primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune haemolytic anaemia (AIHA), diabetes, Pemphigus vulgaris, Hashimoto's thyroiditis, autoimmune inner ear disease myasthenia gravis, pernicious anemia, Addison's disease, dermatomyositis, Sjogren's syndrome, dermatomyositis, multiple sclerosis, Reiter's syndrome, Graves disease, autoimmune hepatitis, familial adenomatous polyposis and ulcerative colitis.
Primary Immune Thrombocytopenia (ITP) is an autoimmune disorder characterized by a low platelet count (thrombocytopenia) of unknown aetiology. The immune system of ITP patients produces antibodies directed against their own platelets. These auto-antibodies form immune-complexes with platelets, which are subsequently recognised by Fc-gamma receptors (FcγRs) expressed on immune cells. This interaction triggers a wide range of responses which includes up-take, processing, antigen presentation as well as initiation of cellular cytotoxicity and release of inflammatory and immune mediators. As a result the platelet count in the blood is decreased and patients start suffering from bruising and potentially fatal spontaneous bleedings (purpura). The reason why the body reacts against its own platelets is currently unknown.
Primary Immune Thrombocytopenia is defined by platelet counts<100,000 μL (Rodeghiero, F. et al. (2009)) and is characterized clinically by an increased tendency to bruise. Clinically, ITP often presents as spontaneous bleeding in individuals with platelet counts of less than 20,000 μL. Subjects with platelet counts<10,000 μL may present with severe cutaneous bleeding, gingival bleeding, epistaxis, haematuria or menorrhagia. Some bleeding risk is present in subjects with platelet counts between 30,000 μL and 50,000 μL depending on the coexisting factors for bleeding (Cines D., (2005)).
Primary Immune Thrombocytopenia affects between 1 and 4 in 10,000 persons in the European Union, which corresponds to 50,000-200,000 persons in total (SuppreMol, 2008)). Children account for about half of all cases. In the United States, the ITP prevalence is estimated between 1 and 2.4 per 10,000 corresponding to up to 72,300 patients in total (Segal, J., (2006)), (Feudjo-Tepie, M., et al., (2008)). Children developing ITP experience acute disease followed by complete spontaneous remission, in most cases. In contrast, more adults will develop chronic ITP; a disease characterized by persistent moderate to severe thrombocytopenia that puts them at risk for bleeding with trauma and can also result in spontaneous haemorrhage of variable severity.
Diagnosis of ITP in adults is based on confirmed thrombocytopenia together with exclusion of other causes for the thrombocytopenia based on physical examination, complete blood count and blood smear results (George, J. et al., (1996)). Assays for anti-platelet antibodies are not sufficiently sensitive or specific to justify their routine use in the diagnosis of ITP (Brighton T. et al., (1996)).
The American Society of Hematology and the British Society for Haematology recommend initial treatment consisting of a full course of steroids or intravenous immunoglobulin. The initial treatment in adults with severe ITP is prednisone, usually at a dose of 1 mg/kg/day for 2 to 4 weeks. Patients who do not adequately respond to first line treatment are candidates for splenectomy. Two thirds of subjects with ITP who undergo splenectomy will achieve a normal platelet count (British Journal of Haematology, 2003)). Although splenectomy is a routine surgical procedure, it is endowed with a 0.8% risk of surgical mortality and a complication rate of 12% (Baccarani, U. et al., (1999)). Overall, the use of splenectomy for ITP is declining.
Subjects who do not respond to the first and second line treatment are classified as having chronic refractory ITP and this happens for 11-35% of ITP subjects. The treatment options available are:
High dose steroids: Oral dexamethasone of short duration in cycles and parenteral methylprednisolone. The response to the latter is faster therefore this is indicated when the platelet count needs to be increased as a priority. This response is transitory and requires maintenance therapy in the form of oral steroids.
High doses of IVIG: A small number of studies have shown that IVIG is effective and raises platelet count faster than steroids in adults with ITP. The doses used varied but 1 mg/kg/day for 2 days was generally recommended (Godeau, B. et al., (2003). The response is usually transitory but the infusions of IVIG can be repeated.
Intravenous anti-D: In one study this therapy has been shown to elevate the platelet counts in 79-90% of adults and lasted up to 3 weeks in 50% of those who responded (Scaradavou, A. et al., (1997)). This treatment is only effective in Rh D-positive non-splenectomised patients.
Vinca alkaloids: Vincristine or vinblastine, given i.v., can cause an increase in platelet count lasting 1-3 weeks. 50% of splenectomised patients respond. This response is sustained in only a small proportion of subjects (Berchtold, P. et al., (1989)).
Danazol: When administered at 200 mg 2-4 times daily for more than 2 months it resulted in a 60% response rate. It can be continued for over a year with less toxicity than long-term steroids. Age, sex, and the status of the spleen influence the responses (Ahn, Y. et al., (1989)).
Immunosuppressive agents: Azathioprine and cyclophosphamide have been successfully used (up to 25% of patients showed sustained response) but are slow acting and need long-term administration. Cyclosporin A can be given alone or with prednisone but carries a substantial risk of serious adverse reactions.
Dapsone: Administered orally for several weeks at a dose of 75 to 100 mg, resulted in a remission in half of the treated subjects but it was less effective in subjects with severe ITP and in splenectomised subjects (Godeau, B. et al., (1997)).
Experimental agents which have been used with good effect include thrombopoietic agents as well as rituximab or mycophenolate mofetil.
Rituximab, romiplostim, and eltrombopag are potential agents that have demonstrated the ability to increase platelet counts in patients with chronic ITP. Quite recently, the thrombopoietin-receptor agonists romiplostim and eltrombopag have been approved by the FDA for the treatment of chronic ITP in splenectomised patients. Romiplostim is also approved in the EU.
Romiplostim, a subcutaneous injectable analog of thrombopoietin, and eltrombopag, an oral non-peptide molecule, are indicated in the US for the treatment of thrombocytopenia in patients with chronic ITP who have an insufficient response to corticosteroids, immunoglobulins, or splenectomy.
Response was seen in splenectomised and non-splenectomised patients, including those who had no sustained benefit from multiple other agents. Further investigations need to be performed to define the risks of long-term use of thrombopoietic stimulating agents and the benefit of these novel agents in comparison to other therapies that provide a durable response off therapy (Burzynski J., (2009)).
Mycophenolate mofetil is a new immunosuppressive agent used in ITP. One study has shown 39% of patients achieved a sustained response. This agent may be a useful component of a combination therapy in patients with refractory ITP (Provan D. et al., (2006)).
In subjects requiring emergency treatment for low platelet count associated with active bleeding, high doses of i.v. corticosteroids or IVIG are indicated and transfusion of random donor platelets may be appropriate.
In spite of these different approaches for treating an autoimmune disease, they still represent a major health impairment and therefore further treatment approaches are needed.